original article renoprotective and …€¦ · după tratarea timp de 6 săptămâni cu he (30 și...

FARMACIA, 2020, Vol. 68, 6

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https://doi.org/10.31925/farmacia.2020.6.19 ORIGINAL ARTICLE

RENOPROTECTIVE AND HEPATOPROTECTIVE EFFECTS OF

HIPPOCRATEA EXCELSA ON METABOLIC SYNDROME IN

FRUCTOSE-FED RATS

ELIZABETH ALEJANDRINA GUZMÁN HERNÁNDEZ 1,2*, SILVANA ANDREA DÍAZ

PORTILLO 1, ÓSCAR CRISTÓBAL VILLAFUERTE ANAYA 1, MARÍA DEL ROSARIO

GONZÁLEZ VALLE 3, JOSÉ DEL CARMEN BENÍTEZ FLORES 3, RUBÉN SAN MIGUEL

CHÁVEZ 4, GLADYS CHIRINO GALINDO 5, LEONARDO DEL VALLE MONDRAGÓN 6,

DAVID SEGURA COBOS 2, GIL ALFONSO MAGOS GUERRERO 7, PEDRO LÓPEZ SÁNCHEZ 1

1Postgraduate Studies and Research Section, Higher School of Medicine, National Polytechnic Institute, Mexico City, 11340,

Mexico 2Medical Surgeon Career, Faculty of Superior Studies Iztacala, National Autonomous University of Mexico, Tlalnepantla,

State of Mexico, 54090, Mexico 3Histology Laboratory, Morphology and Function Unit, Faculty of Superior Studies Iztacala, National Autonomous

University of Mexico, Tlalnepantla, State of Mexico, 54090, Mexico 4Phytochemistry Area, Postgraduate Degree in Botany, Campus Montecillo, Postgraduate College, Km. 36.5 México-Texcoco

Road, Montecillo, Texcoco, State of Mexico, C.P. 56230, Mexico 5Biology Career, Faculty of Superior Studies Iztacala, National Autonomous University of Mexico, Tlalnepantla, State of

Mexico, 54090, Mexico 6Department of Pharmacology, National Institute of Cardiology Ignacio Chávez, Mexico City, C.P. 04510, Mexico 7Department of Pharmacology, Faculty of Medicine, National Autonomous University of Mexico, Coyoacán, Mexico City,

C.P. 04510, Mexico

*corresponding author: [email protected]

Manuscript received: January 2020

Abstract

The metabolic syndrome is associated with the development of chronic kidney disease and liver damage. The aim of this research

was to determine the effect of the ethanol bark extract of Hippocratea excelsa (HE) on high fructose consumption-induced

adverse effects in the kidney and liver of rats. Rats with 20% fructose feeding for 12 weeks showed arterial hypertension, obesity,

dyslipidaemia and developed oxidative stress, proteinuria, the activities of antioxidant enzymes in the renal cortex and liver

were decreased, TGF-1 increased, and kidney and liver damage were observed. After the treatment for 6 weeks with HE (30 and

100 mg/kg bw) renoprotective and hepatoprotective effects in high fructose induced metabolic syndrome in rats, were demonstrated.

Rezumat

Sindromul metabolic este asociat cu dezvoltarea bolilor renale cronice și a afectării hepatice. Scopul acestei cercetări a fost de

a evalua acțiunea extractului etanolic din scoarța de Hippocratea excelsa (HE) asupra efectelor adverse induse de consumul

ridicat de fructoză, la nivel renal și hepatic, la șobolani. Șobolanii cu hrana îmbogățită cu 20% fructoză, timp de 12 săptămâni,

au prezentat hipertensiune arterială, obezitate, dislipidemie și au dezvoltat stres oxidativ, proteinurie. Activitățile enzimelor

antioxidante din cortexul renal și ficat au scăzut, TGF-1 a crescut și au fost observate leziuni la nivelul rinichilor și ficatului.

După tratarea timp de 6 săptămâni cu HE (30 și 100 mg/kgc) s-au observat efecte renoprotectoare și hepatoprotectoare la

șobolanii cărora li s-a indus sindromul metabolic prin consumul ridicat de fructoză.

Keywords: Hippocratea excelsa, kidney disease, metabolic syndrome, liver damage

Introduction

Metabolic syndrome is a serious threat to public health

because it is closely related to the modern lifestyle,

diet plays an important role in growth and development

as a source of nutrition, but the composition of the

diet decides its nutritional status. The modern diet,

especially in Western countries, is rich in carbo-

hydrates such as fructose and sucrose as well as

saturated fat. This increased caloric intake affects

multiple metabolic functions and has been associated

with a higher incidence of the metabolic syndrome [1].

Excess weight and obesity are associated with hemo-

dynamic, structural and histological renal and liver

changes, in addition to metabolic and biochemical

alterations that lead to kidney disease and liver

injury [2].

Combinations of carbohydrate and fat-rich dietary

components have been used in rodents to mimic these


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signs and symptoms of human metabolic syndrome

and his association with renal and liver damage [3].

Hippocratea excelsa HBK. (Hippocrateaceae) (syn.:

Hemiangium excelsum HBK) is a liana native to

Mexico and Central America. The root bark of this

plant, known as “Cancerina”, is used in the Mexican

traditional medicine for the treatment of peptic ulcers,

gastrointestinal infections, skin ailments, kidney

disease, menstruation disorders and as antihypertensive

[4]. Root bark of H. excelsa has been widely studied

in México for its anti-inflammatory, antiparasite and

in vitro anti-tumour effects [5]. H. excelsa, as anti-

inflammatory agent produced a significant inhibition

of carrageenan-induced paw oedema and reduced the

weight of cotton pellet-induced granuloma at doses

of 25 - 100 mg/kg bw [5]. H. excelsa, for its anti-

tumour effect, was used in bioscreening studies to

detect the cytotoxic activity against human tumour

cells in three different extracts (petroleum ether,

ethylacetate and methanol) [5]. In the present study,

we examined the effect of H. excelsa administration on

liver damage and kidney disease, in a high fructose

induced metabolic syndrome rat model.

Materials and Methods

Preparation and identification of the ethanol extract

of H. excelsa

The root bark of H. excelsa was collected on April 2016

at Costa Grande, Guerrero, Mexico and authenticated

by Edith López Villafranco, biologist. A voucher

specimen (2483) has been deposited at the Herbarium

of the Botany Department of the Faculty of Superior

Studies Iztacala, National Autonomous University of

Mexico (UNAM).

For the in vivo evaluation, powered H. excelsa root

bark (3 kg) was extracted twice by maceration with

ethanol (30:l v/w) at room temperature for 72 h, filtered

and evaporated in vacuo (50°C). The dry ethanol

extract was stored at 4°C. The yield of obtaining the

ethanol extract of H. excelsa (HE) was 6.5%.

Phytochemical profiling

For the chromatographic analysis of HE it was used a

high-resolution liquid chromatograph Hewlett Packard

Mod. 1100, equipped with an automatic injector (Agilent

Technologies Mod. 1200), a diode array detector

(Hewlett Packard Mod. 1100) and a quaternary pump

HP Mod. 1100.

Chromatography for the analysis of phenolic acids in

HE was performed on a nucleosil 100A 125 x 4 mm

column, adjusted to 30º, using a linear gradient of 1

mL/min of water (pH 2.5 with trifluoroacetic acid)

(Solution A) and acetonitrile (solution B). Initially, (0

to 0.1 min) 85% solution A and 15% solution B, (0.1

to 20 min) 65% solution A and 35% solution B and

(20 to 23 min) 65% solution A and 35% solution B;

injection volume: 20 µL; the phenolic acids were

detected at 280 nm.

For the flavonoids in HE, the chromatography was

performed on a Hypersil ODS 100A column of 123 ×

4.0 mm, adjusted to 30º. The system was operated

with gradient elution with solution A: water (pH 2.5)

with trifluoroacetic acid and solution B: acetonitrile,

with a linear gradient of 1 mL/min. Initially, (0 to 0.1

min) 85% solution A and 15% solution B, (0.1 to 20

min) 65% solution A and 35% solution B and (20

to 25 min) 65% solution A and 35% solution B;

injection volume: 20 μL; flavonoids were detected

at 254, 316 and 365 nm.

The terpenoid analysis was performed with a ZORBAX

Eclipse XDB-C8 column (4 mm × 125 mm, 5 μm).

The major constituents were separated with gradient

mobile phase; and the flow was adjusted to 1 mL/

min for 21 min; that consists of water 20% and aceto-

nitrile 80%; the detection wavelength of 215 and 220

nm; 20 µL injection volume.

In vitro antioxidant capacity

Determination of total phenolic content (TPC). The

determination of TPC of the ethanol extract of H.

excelsa was performed by Folin-Ciocalteu method

with little modifications, using gallic acid as a standard

phenolic compound [6]. The extract was diluted with

distilled water to a known concentration in order to

obtain the readings within the standard curve range

of 0.0 to 600.0 µg of gallic acid/mL. A volume of

250 µL of diluted extract or gallic acid solution was

mixed with 1 mL of distilled water in a test tube

followed by the addition of 250 µL of Folin-Ciocalteu

reagent. The samples were mixed and then allowed

to stand for 5 min at room temperature in order to

allow complete reaction with Folin-Ciocalteu reagent.

Then 2.5 mL of 7% sodium carbonate aqueous solution

was added and the final volume was made up to 6 mL

with distilled water. After incubating the samples for

90 min at room temperature, the absorbance of the

resulting blue colour solution was measured at 760 nm

using a spectrophotometer. The result was expressed

as mg of gallic acid equivalents (GAE)/g extract by

using an equation that was obtained from standard

gallic acid curve. All the experiment was conducted

in triplicate.

DPPH radical scavenging assay. The DPPH assay

was carried out as described by Hsu et al. with some

modifications [7]. A volume of 1.5 mL of 0.1 mmol/L

DPPH solution was mixed with 1.5 mL of various

concentrations (10 to 500 µg/mL) of bark extract. The

mixture was shaken vigorously and incubated at room

temperature for 30 min in the dark. The reduction

of the DPPH free radical was measured by reading

the absorbance at 517 nm by a spectrophotometer.

The solution with DPPH and methanol was used as

negative control. The experiment was replicated in

three independent assays. Quercetin was used as

positive control. Inhibition of DPPH free radical in

percentage was calculated by the formula:


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DPPH radical scavenging activity (%) = (Acontrol -

Atest)/Acontrol × 100,

where, Acontrol is the absorbance of the negative control

and Atest is the absorbance of samples. The antioxidant

activity of each sample was expressed in terms of

IC50 (micromolar concentration required to inhibit

DPPH radical formation by 50%), calculated from the

graph after plotting inhibition percentage against the

extract concentration.

ABTS radical scavenging assay. In order to assess

the ABTS radical scavenging assay, the method of Re

et al. was adapted [8]. The stock solutions included

7 mmol/L ABTS solution and 2.4 mmol/L potassium

persulfate solution. The working solution was then

prepared by mixing the two stock solutions in equal

quantities and allowing them to react for 12 h at room

temperature in the dark. The resulting solution was

then diluted by mixing 1 mL of freshly prepared ABTS

solution to obtain an absorbance of (0.706 ± 0.001)

units at 734 nm using the spectrophotometer. Fresh

ABTS solution was prepared for each assay. The

plant extract (1 mL) was allowed to react with 2.5

mL of the ABTS solution and the absorbance was

registered at 734 nm after 7 min using a spectrophoto-

meter. The ABTS scavenging capacity of the extract

was compared with that of Trolox and the percentage

inhibition was calculated as:

ABTS radical scavenging activity (%) = (Acontrol -

Atest)/Acontrol × 100,

where Acontrol is the absorbance of ABTS radical +

methanol; Atest is the absorbance of ABTS radical +

sample extract/standard.

Reducing Power Assay (FRAP). For FRAP (ferric

reducing antioxidant power) assay, extract/fraction

solution (0.1 mL) was added to reagent (2 mL) in

acetate buffer (0.3 M, pH 3.6), 2,4,6-tris(2-pyridyl)-

s-triazine (TPTZ) (10 mM) in 40 mM HCl and ferric

chloride (20 mM) in a final ratio of 10:1:1 (v/v/v).

Then, the absorbance at 593 nm was read after 30

min of incubation at room temperature. Similarly, a

blank sample (prepared in the same manner, but without

the extract) was prepared. Millimoles of Trolox

equivalents per gram of ethanolic extract H. excelsa

(TEs/g extract) were the measurement unit [9].

Ethical consideration and animals used

The study was submitted to the Animal Use Ethics

Committee of Faculty of Superior Studies Iztacala,

UNAM. It was approved under Protocol No. CE/FESI/

102016/1110). The handling of the laboratory animals

followed the rules for the Care and use of laboratory

animals of the Official Mexican Rule (NOM-062-

ZOO-1999, revised in 2001); the International Guide

for Caring and Use of Laboratory Animals NRC

2002; all procedures and experimental protocols are

in compliance with the European Communities Council

Directive of 24 November 1986 (86/609/EEC).

Thirty male Wistar rats were used, each with a weight

of around 200 - 250 g. During the study, the animals

were housed in individual stainless steel metabolic

cages, measuring 60 cm × 50 cm × 22 cm. They

were kept in an air-conditioned environment, with a

temperature of 25 ± 3°C, and a humidity of 50 ±

10%, a photoperiod of 12 h of light and dark, and

they were fed with standard balanced food ratios for

rodents and water ad libitum.

Induction of metabolic syndrome

The control diet (2018s Teklad Global 18% protein

rodent diet from Harlan Laboratories) contained

proteins (18.6%), carbohydrates (44.2%) and fat

(6.2%). Chow and drinking water with 20% fructose

were elaborated [10]. Rats were initially divided into

two groups: control group (n = 6) and fructose feed

(F) group (n = 24), and treated for 12 weeks under

the next conditions: the control group with regular

chow and drinking water, and the fructose fed group

with 20% fructose in chow and drinking water.

Experimental design

After 12 weeks of fructose treatment, the rats were

randomly divided into four groups (n = 6), were

maintained under initial diet conditions and treatments,

and were orally administrated for 6 weeks, as follows:

Control group; Metabolic syndrome (F); Metabolic

syndrome treated with losartan 10 mg/kg bw (F + Los);

Metabolic syndrome group treated with vitamin E

500 mg/kg bw (F + Vit E); Metabolic syndrome treated

with ethanol extract of H. excelsa (HE): 30 mg/kg

bw and 100 mg/kg bw (F + HE 30 and F + HE 100).

The HE doses used were based on the toxicity study,

the lowest dose that did not present toxic effect (30

mg/kg bw and 100 mg/kg bw) were used.

After 6 weeks of treatment, rats were kept in metabolic

cages, for evaluating water intake, food intake, and

urinary volume at 24 hours; urine samples were used

for protein concentration measurement by Bradford

method (Bio-Rad) [11].

Blood pressure

Systolic arterial blood pressure (SBP) was measured

noninvasively using a tail-cuff computer-aided monitoring

device (Automatic Blood Pressure Computer, Model

LE 5007; Letica Scientific Instruments, Barcelona,

Spain) using the procedures described [12], at the

beginning (0 week), middle (12 weeks) and end (6

weeks) of the experiment.

Biochemical analyses

Blood concentrations of glucose, total cholesterol and

triglycerides were measured using an Accutrend Sensor

glucometer (Roche), at the beginning (0 week), middle

(12 weeks) and end (6 weeks) of the experiment.

On the 6th week of treatment, the blood was collected

(3 mL) for the biochemical assessment. High-density

lipoprotein (HDL) (Spinreact, Cat. 1001097) and LDLc

(Spinreact, Cat. 41023) cholesterol levels, aspartate

aminotransferase (AST) (Spinreact, Cat. 12531) and

alanine aminotransferase (ALT) (Spinreact, Cat. 12533)


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were measured using commercially available kits

following the manufacturer´s protocol.

Very-low-density lipoprotein cholesterol (VLDLc)

was calculated using the formula: VLDLc = 0.2 x

TAG. Cardiac index was calculated as TC/HDLc.

Atherogenic index was calculated TC-HDLc/HDLc

and coronary artery index was calculated as LDLc/

HDLc [13].

The plasma concentration of angiotensin II, angiotensin

(1-7), nitric oxide and endothelin were measured by

capillary zone electrophoresis. Plasma was deproteinized

with methanol 10:1 (v:v) and centrifuged at 16,000

x g for 10 min at 4°C (Sorvall RC-28S, rotor SS34;

DuPont, Newtown, CT, USA). The pellet was discarded,

and supernatant was deproteinized by the addition of

20% trichloroacetic acid, homogenized and centrifuged

at 16, 000 x g for 10 min at 4°C. The supernatant was

filtered through a 0.22 µm nitrocellulose membrane

filter (Millipore, Billerica, MA, USA) and diluted

1:10 with 0.1 M NaOH. The sample (2 mL) was then

passed through a Sep-Pak Classic C-18 cartridge (Waters

Corporation, Milford, MA, USA) as described by

[10, 11]. These experiments were performed using

a Beckman Coulter (Fullerton, CA, USA) P/ACETM

MDQ Capillary Electrophoresis System equipped

with PDA and controlled by means of the P/ACE

MDQ Capillary Electrophoresis System software

(version 7.0; Beckman Coulter Inc., Fullerton, CA,

USA) [14, 15].

Histopathological analysis

At the end of the treatments, animals were weighted

and anesthetized with sodium pentobarbital (45 mg/

kg bw, intraperitoneally). The mass of each organ

and tissue was measured: kidneys, retroperitoneal

adipose tissue and omental adipose tissue. The histo-

pathological analysis of the organs was realized

following the technique previously described [16],

kidneys and livers were placed in paraformaldehyde

4%, were dehydrated through ethanol graded series,

embedded in paraffin, sectioned in 5 µm thick slices,

mounted on glass slides and stained with haematoxylin-

eosin. Sections of renal cortex further subjected to

morphometric analysis 10 adjacent non-overlapping

fields from each group were randomly chosen and

examined by the light microscope (Leica DMD 108)

using a magnification of 40X.

Western blotting assessment

The kidneys were perfused and rapidly removed. The

cortex was isolated before western blotting and enzyme

activity measurements. The renal tissue was homogenized

in 100 mM Tris (hydroxymethyl-aminomethane-tris-

hydrochloride, Sigma, St Louis, MO, USA), pH 7.4,

incubated with a protease-inhibitor cocktail (Complete

Mini, EDTA-free protease inhibitor cocktail, Roche,

Germany) and centrifuged at 10,000 x g for 10 min to

remove insoluble debris. Aliquots containing 80 μg

of protein were separated by reducing 10% (w/v)

polyacrylamide gel electrophoresis and electroblotted

to polyvinylidene difluoride membranes. Coloured

molecular weight standards (GE Healthcare, Piscataway,

NJ, USA) were run simultaneously. Membranes were

blocked for 2 h in 5% (w/v) non-fat milk and incubated

overnight in the presence of the corresponding anti-

bodies (rabbit polyclonal antibody to AT1R, mouse

monoclonal antibodies to transforming growth factor

beta 1 (TGF-β1) and β-actin (Santa Cruz Biotechnology

Inc., Santa Cruz, California, USA)) (1:1000 dilution)

in 5% (w/v) BSA in phosphate-buffered saline (PBS)

containing 0.1% (v/v) Tween 20, at 4°C. After incubation

for 2 h at room temperature in the presence of the

corresponding horseradish-peroxidase-conjugated

secondary antibodies (Santa Cruz Biotechnology Inc.,

Santa Cruz, California, USA) (1:1000 dilution).

Complexes were visualized by chemiluminescence

detection. Films were scanned, and densitometric

analysis was performed using the software Multi Gauge,

Fuji Film Science, Lab2003 (Fuji Photo Film Co.,

LTD).

Evaluation of oxidative stress

Renal and liver tissue catalase (CAT) activity was

assayed at 25ºC, method which is based on the

disappearance of H2O2 from a solution containing

30 mmol/L H2O2 in 10 mmol/L potassium phosphate

buffer (pH 7) at 240 nm [17]. The glutathione peroxidase

(GPx) activity was assayed by a previously described

method [18]. Results were expressed as UI/mg protein.

Superoxide dismutase (SOD) activity in renal cortical

homogenates was measured by a competitive inhibition

assay using xanthine–xanthine oxidase system to reduce

NBT [19]. Results were expressed as UI/mg protein.

Statistical Analysis

The data represent the mean ± SEM from 6 rats per

treatment. All statistical analyses were performed

using GraphPad Prism 5.00 (GraphPad Software, La

Jolla, California, USA). C, F, F + Los, F + Vit E, F +

HE 30 and F + HE 100 groups were tested for effects

of diet, treatment, and their interactions by two-

factor analysis of variance (ANOVA). When the

interaction and/or the main effects were significant,

means were compared using Tukey´s multiple comparison

post hoc test.

Results and Discussion

MS is a progressive health disorder associated with

different risk factors, including hyperglycaemia,

dyslipidaemia, hypertension and obesity, and that

predisposes to cardio-renal dysfunction [20-22].

The chemical composition of the extract of

Hippocratea excelsa is presented in Table I.

Fructose is a highly lipogenic sugar [22]; the

administration of 20% fructose to rats for 12 weeks

induced the classic symptoms of MS; blood tri-

glycerides (TGs), body weight gain, body mass index

and abdominal circumference increased, correlated

with the increase of weight of total abdominal adipose


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tissue (mesentery, retroperitoneal and epididymal fat)

compared to animals with normal diet (Table II and III).

Hence, we have used this fructose induced metabolic

syndrome rat model [10] to investigate whether the

oral administration of ethanol extract of H. excelsa

(HE) for 6 weeks can reverse the alterations in liver

and renal parameters.

Table I

Phytochemical constituents of the ethanol extract of Hippocratea excelsa

Retention time (min) Area (mAU * s) Identification Quantification (%)

2.502 85.00604 gallic acid 0.045

4.420 24.63361 chlorogenic acid 0.16

5.491 683.4551 vanillinic acid 0.11

6.703 500.3157 caffeic acid 0.22

9.416 28.79682 ferulic acid 0.05

10.020 43.70384 p-cumaric 0.03

4.260 6500.446 oleanolic acid 1.6

2.539 127.2414 ursolic acid 0.26

6.386 110.4542 α-amyrin 9.3

6.718 23.818 phloridzin 0.017

12.275 9.0686 naringenin 0.015

21.428 26.4578 galangin 0.013

Table II

Effect of fructose feeding over body, plasma, and urinary parameters

Variables Control Fructose

Initial body weight (g) 231 ± 4.37 225 ± 2.99

Body weight at 12 weeks (g) 398 ± 19 436 ± 10 *

Body weight gained (1 - 12 weeks) (%) 7.2 ± 1.74 9.4 ± 1.2

Body mass index (g/cm3) 0.89 ± 0.04 0.88 ± 0.05

Abdominal circumference (cm) 17 ± 0.58 22 ± 0.085 *

Lee index 0.28 ± 0.01 0.34 ± 0.01 *

Plasma glucose (mmol/L) 4.64 ± 0.48 4.89 ± 0.24

Plasma triglycerides (mmol/L) 1.044 ± 0.25 2.72 ± 0.46 *

Plasma cholesterol (mmol/L) 2.6 ± 4 2.7 ± 5

Urinary volume (mL) 7 ± 2 27 ± 6 *

Food intake (g/day) 20 ± 2 48 ± 3 *

Water intake (mL/day) 35 ± 9 79 ± 13 *

Urine protein excretion (mg/24 h) 27 ± 4 115 ± 24 *

Systolic blood pressure (mmHg) 116 ± 15 140 ± 5 * Mean ± SEM; n = 6 for control group and n = 30 for fructose fed group. Statistically significant compared with the control group; * = p < 0.05

Table III

Effect of HE treatment on metabolic variables in fructose fed rats

Variables Control F Los Vit E HE 30 HE 100

Food intake (g/day) 42 ± 4 14 ± 5 15 ± 2 15 ± 4 16 ± 4 18 ± 4

Water intake (mL/day) 42 ± 4 70 ± 5 66 ± 8 87 ± 8 69 ± 9 76 ± 10

Body weight at 18 weeks (g) 434 ± 20 572 ± 19 * 505 ± 16 491 ± 31 522 ± 16 551 ± 16 *

Body weight gained (12 - 18

weeks) (%)

14 ± 3 30 ± 2 * 15 ± 2 & 16 ± 2 & 16 ± 2 & 19 ± 2 &

Visceral adiposity index (%) 2.53 ± 0.46 4.93 ± 0.57 * 3.95 ± 0.44 * 4.37 ± 0.68 4.72 ± 1.13 3.86 ± 0.30 *

Body mass index (g/cm3) 0.73 ± 0.02 1.05 ± 0.11 * 0.76 ± 0.03 & 0.74 ± 0.05 & 0.75 ± 0.02 & 0.77 ± 0.02 &

Abdominal circumference (cm) 19 ± 0.37 22 ± 0.6 * 20 ± 0.32 19 ± 0.5 20± 0.48 21 ± 0.48

Lee index 0.31 ± 0.01 0.36 ± 0.02 * 0.31 ± 0.01 & 0.30 ± 0.008 & 0.30 ± 0.003 & 0.30 ± 0.004 &

Tissue wet weight (mg/mm)

Retroperitoneal adipose

tissue

152 ± 32 326 ± 47 * 135 ± 20 & 196 ± 39 & 148 ± 12 & 205 ± 45 &

Omental adipose tissue 144 ± 34 255 ± 40 * 184 ± 47 138 ± 23 & 150 ± 27 & 188 ± 67

Plasma glucose (mmol/L) 4.42 ± 0.30 5.25 ± 0.23 5.55 ± 0.24 5.06 ± 0.12 5.07 ± 0.04 5.6 ± 0.22

Plasma triglycerides (mmol/L) 1.18 ± 0.19 2.62 ± 0.58 * 3.4 ± 0.28 * 2.53 ± 0.34 * 2.57 ± 0.12 * 3.17 ± 0.55 *

Plasma cholesterol (mmol/L) 2.0 ± 3.7 2.0 ± 4 2.34 ± 6 2.5 ± 5 2.2 ± 4 2.3 ± 8

AST (UI/L) 6.4 ± 3 26 ± 2 * 18 ± 3 & 14 ± 2 & 16 ± 3 & 8.6 ± 2 &

ALT (UI/L) 9.3 ± 2 37 ± 3 * 25 ± 4 & 19 ± 4 & 21 ± 3 & 17 ± 3 & F = fructose fed rats; Los = F + losartan 10 mg/kg bw; Vit E = F + vitamin E 500 mg/kg bw; HE 30 = F + HE 30 mg/kg bw;

HE 100 = F + HE 100 m/kg bw; n = 6; * = p < 0.05 control vs. treatment, & = p < 0.05 fructose fed rats vs. treatment


1111

The fructose fed groups treated with losartan, vitamin

E and HE showed lower Lee index, blood triglycerides,

weight of retroperitoneal adipose tissue and omental

adipose tissue compared with fructose fed animals

(Table III). Losartan treatment significantly elevated

the serum concentrations of total adiponectin in patients

with essential hypertension [23]. In rats, losartan reduced

leptin concentration in both losartan and high fat diet

and losartan groups [24]. Leptin is released from

adipose tissues into the blood stream and regulates

appetite, feeding and energy expenditure [24]. Liver

functions were evaluated in the rat by determining the

serum concentrations of ALT and AST. Activities of

AST and ALT are most commonly used as biochemical

markers for liver damage. Since these enzymes are

cytoplasmic in nature, upon liver injury these enzymes

enter into the circulatory system due to altered

permeability of membrane [25]. As shown in Table III,

serum levels of AST and ALT were significantly

increased after 18-weeks of high fructose feeding (p

< 0.05) [26]; HE significantly prevented high

fructose induced elevation of AST and ALT, indicating

the hepatoprotective activity of HE. Oral treatment

with α-amyrin (20 mg/kg bw), a pentacyclic tri-

terpenoid that is a component of HE, attenuated the

increase of AST and ALT enzymes activities in a rat

model of CCl4-induced hepatic oxidative stress and a

subsequent recovery towards normalization of these

enzymes [27].

Figure 1.

Chromatographic profile of root bark ethanol

extract of H. excelsa

The chromatographic profile of HE revealed the

presence of several phytoconstituents (Figure 1 and

Table I): α-amyrin, oleanolic acid, ursolic acid, caffeic

acid, and chlorogenic acid. The components of HE: α-

amyrin and oleanolic acid, cause the effects attributed

to H. excelsa. Alpha-amyrin treatment prevented the

increase in blood triglycerides [28], weight of retro-

peritoneal adipose tissue and omental adipose tissue

[29], observed effects in HE treated groups in this

work (Table III).

Figure 2.

Lipid profile: (a) LDL, (b) HDL, (c) VLDL and (d)

atherogenic, (e) cardiac and (f) coronary artery

indexes in rats with fructose induced metabolic

syndrome after six weeks of administration of

ethanol extract of H. excelsa (HE) * = p < 0.05 control vs. treatment; & = p < 0.05 MetS vs.

treatment: Control, fructose fed rats (F), F + losartan (Los),

F + vitamin E (Vit E), F + HE 30 mg/kg bw (HE 30) and

F + HE 100 mg/kg bw (HE 100)

Impact of treatment with HE on lipid profile

Dietary fructose in the liver is rapidly taken up by

the liver, where it can be converted to glycerol-3-

phosphate, favouring the esterification of unbound

fatty acids to form TGs [30]. Hypertriglyceridemia

occurs following 12 weeks of consumption of fructose,

marked by elevated levels of plasma triglycerides;

VLDLc and LDLc in rats with MS increased significantly

(p < 0.05) compared with the control (Figures 2a

and 2c). Conversely, the HDLc significantly lowered

(Figure 2b); these alterations are features of athero-

sclerosis and cardiovascular disease [31]. These changes


1112

in rats with MS were attenuated by HE compared

with losartan and vitamin E treated groups (Figures 2a

and 2c). Furthermore, calculated atherogenic, cardiac

and coronary artery indexes were increased in rats

with MS compared with the control (Figure 2d);

administration of losartan, vitamin E and HE in fructose

fed rats, reversed the increases in these indexes

(Figures 2d, 2e and 2f). The attenuation of decrease

of HDLc by HE (Figure 2b) shows its ability to

prevent the development of atherosclerosis [32].

Oleanolic acid reduced serum triglycerides, total

cholesterol, and LDL cholesterol [33]. Chlorogenic

acid reduced total cholesterol and LDL-cholesterol,

increased HDL cholesterol, and improved both the

atherogenic index and the cardiac risk factor, to inhibit

fatty acid synthase and hydroxyl methyl glutaryl

coenzyme A reductase [34]. Alpha-amyrin reduced

serum triglycerides, total cholesterol, LDL-cholesterol,

atherogenic index, and increased HDL cholesterol [28].

Antihypertensive effect of HE

In the fructose-fed rats, the systolic blood pressure

(SBP) was increased compared with the control (148 ±

3 mmHg for MS group compared with 106 ± 2 mmHg

in control group); this increase was prevented by

losartan (115 ± 3 mmHg) and HE 30 mg/kg bw (119 ± 3

mmHg). Increase in SBP after chronic fructose feeding

was partially abolished with HE 100 mg/kg bw treatment

(130 ± 6 mmHg) (Figure 3).

Figure 3.

Effect of the ethanol extract of H. excelsa (HE) on

systolic blood pressure of fructose fed rats (F) 6

weeks after the establishment of metabolic

syndrome Values are the mean ± SEM (n = 6); * = p < 0.05 control

vs. treatment; & = p < 0.05 F vs. treatment: F + losartan

(Los), F + vitamin E (Vit E), F + ethanolic extract of H.

excelsa (HE) 30 mg/kg bw (HE 30) and F + ethanolic

extract of H. excelsa 100

Figure 4.

Effect of ethanol extract of H. excelsa (HE) on the plasma level of (a) angiotensin II, (b) angiotensin 1-7, (c)

nitric oxide and (d) endothelin in fructose fed rats (F) All values are represented as mean ± SEM; n = 6; * = p < 0.05 control vs. treatment; & = p < 0.05 F vs. treatment: F +

losartan (Los); # = p < 0.05 Los vs. treatment; + = p < 0.05 Vitamin E (Vit E) vs. treatment, F + HE 30 and HE 100

The administration of fructose decreased the systemic

synthesis of vasodilator NO and Ang 1-7 with an

increase of vasoconstrictor endothelin 1 (Figures 4a -

4c); this effect was associated with increase in the


1113

SBP at 12 weeks of fructose fed, as has been shown

by other authors [10, 35]. The compression of kidney

by the adipose tissue around it causes activation of

the RAS [36]. The activation of RAS causes retention

of sodium and water by angiotensin II and leads to

the development of hypertension. The pharmacological

inhibition of RAS reduced blood pressure to about

50% to 60% (Figure 3) [32-35, 37]. The administration

of HE prevented the increase of SBP induced by

administration of fructose, so the release of angiotensin

1-7 and release of NO seems to be one mechanism of

action in the anti-hypertensive effect of HE (Figure 4d).

Histopathological study

In this study, we found that fructose feeding conducted

to kidney hypercellularity (gh), which is an indicator

of proliferative glomerulonephritis associated with

degenerative changes, atrophy characterized by decrease

in kidney size, number of renal corpuscles per field

and thickness of the cortex, necrosis, thyroidization

and protein deposits located in the proximal convoluted

tubule (TCP) and in the space of the Bowman's capsule

(Figures 5a and 5d).

Pathologically, kidney damage is characterized by a

number of structural changes of kidney cells including

a decreased GFR that can lead to the development

of glomerulosclerosis and tubulointerstitial fibrosis

[38, 39]. Distortion in the architecture of the cortex

and medulla and the significant reduction of the

glomerulus diameter suggest sclerosis in the MS of

the current study. All these events observed in fructose

fed rats were partially ameliorated by treatment with

HE in doses of 30 and 100 mg/kg bw and renal

corpuscles showed a diffuse mild hypercellularity

(Figures 6e and 6f).

Figure 5.

Photomicrographs (40X magnification) showing histopathological changes in different groups, (a) control. Note

that the group treated with fructose (b) is the one that presents the most notorious changes, in this group, the

changes were diagnosed as moderate diffuse extension proliferative glomerulonephritis, hypercellularity (Gh),

protein deposits (pd) in the Bowman capsule space, obliterated capillary lumen. In the rest of the groups, lighter

and multifocal changes were identified. (c) In F + losartan treated group glomerulus only presents

hypercellularity (Gh) and the capillary lumen is not obliterated. (d) In F + vitamin E treated group, glomeruli

present hypercellularity (GH), multifocal extension. (e) and (f) In F + ethanolic extract of Hippocratea excelsa

(HE) treated group, the kidney has an almost normal appearance


1114

Figure 6.

Photomicrographs (40X magnification) of contoured tubules in the different groups. Control group (a). Note that

in the group treated with fructose (b), the most affected tubules correspond to the distal tubules (TCDnx), in

them we find multifocal changes that consist of dilatation that is due to thyroidization, tubular atrophy (Ta) and

necrosis. In the group treated with F + vitamin E (d), tubules with degenerative and necrotic changes of

multifocal distribution are identified. In the fructose groups treated with losartan (c), HE 30 and HE 100 (e and

f), the tubules only show the loss of microvilli, but the tubular arrangement and cellular vitality is maintained

(Cn); in them, only some cells suggest necrosis (Cn)

At the hepatic level, the MS group exhibited cellular

degeneration, massive fatty changes, cytoplasmic

vacuolation and the loss of cellular boundaries (Figure

7b). The liver displayed near normal appearance with

well-preserved cytoplasm and prominent nuclei (Figures

7e and 7f); renoprotective and hepatoprotective effect

of HE was demonstrated in an experimental model

of metabolic syndrome on rats.

We found that the fructose-treated rats showed

renal dysfunctions such as reduced kidney weight,

diminished number of renal corpuscles per field

and proteinuria (Table IV).

Table IV

Effect of ethanol extract of H. excelsa (HE) over renal parameters in fructose fed rats

Variables Control F Los Vit E HE 30 HE 100

Kidney weight (g) 1.32 ± 0.035 1.21 ± 0.03 * 1.45 ± 0.04 1.3 ± 0.07 1.32 ± 0.024 1.28 ± 0.03

Rat body weight ratio (mg/g) 2.79 ± 0.10 2 ± 0.086 * 2.74 ± 0.15 & 2.59 ± 0.17 & 2.54 ± 0.09 & 2.38 ± 0.04 &

Thickness of the cortex (µm) 2721 ± 50 2185 ± 90 * 2355 ± 22 & 1946 ± 103 * 2415 ± 39 & 2453 ± 33 &

Number of renal corpuscles per

field (10X)

9 ± 0.4 5.7 ± 1.0 * 7.5 ± 0.85 & 4.5 ± 0.5 * 8 ± 1.4 & 7.5 ± 0.95 &

Proteinuria (mg/24 h) 17 ± 6 89 ± 14 * 32 ± 2 & 31 ± 4 & 37 ± 8 & 31 ± 5 &

F = rats with fructose induced metabolic syndrome; Los = F + losartan; Vit E = F + vitamin E; HE 30 = F + HE 30; HE 100 = F + HE 100; n

= 6; * = p < 0.05 control vs. treatment; & = p < 0.05 F vs. treatment


1115

Figure 7.

Representative haematoxylin and eosin (H&E) staining photos of liver tissue (40X magnification) showing

histopathological changes in different groups: (A) control; (B) metabolic syndrome (F); (C) F + losartan treated

group, (D) In F + vitamin E treated group, (E and F) In F + ethanolic extract of Hippocratea excelsa (HE) treated group

Figure 8.

Effect of ethanol extract of H. excelsa (HE) on the expression of (a) AT1R and (b) TGF-β1 proteins in renal

cortex of rats with fructose induced metabolic syndrome (F) F = fructose; Los = F + losartan; Vit E = F + vitamin E; HE 30 = F + HE 30; HE 100 = F + HE 100

Values are expressed as mean ± SEM; n = 5; * = p < 0.05 control vs. treatment; & = p < 0.05 F vs. treatment


1116

These findings are in line with previous studies

demonstrating that high fructose resulted in proteinuria

[13, 34]. The overexpression of TGF-β1 in the rat

glomeruli induces proteinuria [40]. However, the

parameters of kidney function in the fructose fed

rats treated with H. excelsa were comparable to those

of the control rats; these results demonstrate that H.

excelsa slowed the progression of functional and

structural damage to the kidney in fructose-fed rats.

Inappropriate activation of renin-angiotensin aldosterone

system (RAS) is a pathophysiologic factor in the link

between hypertension and metabolic syndrome [41];

the profibrogenic cytokine TGF-β1 participates in

kidney damage in high fructose induced MS [42-44].

In this study, it was found an elevated expression of

AT1R and TGF-β1 in MS compared to the lean controls

(Figures 8a and 8b). Conversely, HE treatment did

not change the expression of AT1R, (Figure 8a) and

decreased TGF-β1 protein expression (Figure 8b).

It has been shown that local RAS is significantly up-

regulated during liver fibrosis where angiotensin II

stimulates contraction and proliferation of the activated

hepatic stellate cells and increases the expression of

TGF-β1 through angiotensin II type 1 receptors [45].

The present results showed that losartan and extract of

bark of H. excelsa treatments significantly alleviated

the histological injury of liver, displaying near normal

appearance with well-preserved cytoplasm and prominent

nuclei (Figure 7); hepatoprotective effect of HE was

demonstrated in an experimental model of metabolic

syndrome on rats [46].

Antioxidant capacity in vitro

Other mechanisms involved in renal and liver damage

are concerning oxidative stress. Fructose consumption

increases levels of lipid peroxides and decreases

activities of antioxidant enzymes in the kidney and

liver [47, 48]. High fructose produces reactive oxygen

species (ROS) in vitro and in vivo. In this study was

determined the antioxidant activity in vivo and in

vitro of the ethanol extract H. excelsa.

In the present study, H. excelsa bark extract possessed

high phenolic contents (286.4 mg GAE/g of extract),

was calculated using the standard curve of gallic acid

(standard curve equation: Y = 11.747x + 0.0262,

R2 = 0.998). Other plant that showed relevant anti-

oxidant and medicinal properties is Buddleja cordata;

the methanol extract of B. cordata showed 177.13 ±

1.97 mgEq gallic acid/g, which presented 17.71%

phenolic compounds in the extract; B. cordata showed

antioxidant and neuroprotective effects in the 1-

methyl-4-phenylpyridinium Parkinson disease rat

model [49, 50].

DPPH radical scavenging assay

It is well known that the antioxidant activity of plant

extracts containing polyphenol components is due

to the capacity to be donors of hydrogen atoms or

electrons and to capture the free radicals [51]. In

the present study, H. excelsa ethanolic bark extract

showed a significant effect in inhibiting DPPH, reaching

up to 88% at concentration of 50 µg/mL, showing a

dose response curve of DPPH radical scavenging

activity of H. excelsa compared with standard quercetin.

The IC50 value of H. excelsa extract was 18.05 µg/mL

while the IC50 value of standard antioxidant quercetin

was 5.3 µg/mL. The DPPH assay is one of the most

widely used methods for screening the antioxidant

activity of plant extracts. The antioxidant plant, B.

cordata showed an IC50 value of 64.19 ± 2.09 µg/

mL [51].

ABTS radical scavenging activity

The ethanolic bark extract of H. excelsa were fast and

effective scavengers of the ABTS radical and this

activity was comparable to that of BHT. It exhibited

potent scavenging effects against ABTS with an IC50

value of 21.73 µg/mL almost equivalent to that of

standard Trolox (IC50 value 5.3 µg/mL) [48]. The

percentage of inhibition was 99% for the bark extract

at 70 µg/mL concentration. Another root extract with

antioxidant and hepatoprotective properties, the extract

of Pueraria thunbergiana Benth. showed an IC50

value of 138.0 ± 2.7 µg/mL [53].

The reducing power of Fe2+ by the tested plant was

evaluated. The radical scavenging activity of the plant

extract showed a concentration-dependent reducing

power of 379.23 µg/ET/g/extract, compared to standard

Trolox [49].

Antioxidant enzymes

Oxidative stress is a well-recognized phenomenon

playing an important role in the pathogenesis of

endothelial dysfunction, hypertension, inflammation

and atherosclerotic cardiovascular disease. It is defined

as an impaired balance between free radical production

and endogenous antioxidant capacity, resulting in the

accumulation of oxidative products [13].

The SOD, catalase and GPx activities in renal cortex

and liver were reduced in fructose fed rats compared

with control group (Figures 9a and 10a); administration

of the ethanol bark extract of H. excelsa effectively

prevented the decrease of SOD, CAT, and GPx (p <

0.05, respectively).

These results indicated that ethanol extract of bark

of H. excelsa exerted protective effects against kidney

and hepatic injury induced by high fructose diet, at

least in part, through decreasing oxidative stress

(Figures 9b and 9c); (Figures 10b and 10c), through

enhancing ROS-detoxifying enzymes, possibly by

activating redox transcription factors as nuclear factor

erythroid 2 (Nrf2), perhaps by effect of oleanolic acid

[54]. It has been shown that oleanolic acid inhibited

oxidative stress and activated heme oxygenase 1

(HO-1)/Nrf2 [54].


1117

Figure 9.

Effects of ethanolic extract of H. excelsa (HE) on antioxidant enzymes in fructose induced metabolic syndrome

rats (F). Plasma total antioxidant activity (a), renal cortical catalase (CAT) (b), superoxide dismutase (SOD) (c)

and glutathione peroxidase activities (GPx) (d) Los = F + losartan; Vit E = F + vitamin E; HE 30 = F + HE 30; HE 100 = F + HE 100

All values are represented as mean ± SEM; n = 5; * = p < 0.05 control vs. treatment; & = p < 0.05 F vs. treatment

Figure 10.

Effects of ethanolic extract of H. excelsa (HE) on antioxidant enzymes in fructose induced metabolic syndrome

rats (F). Hepatic catalase (CAT) (b), superoxide dismutase (SOD) (c) and glutathione peroxidase activities (GPx)

(d) Los = F + losartan; Vit E = F + vitamin E; HE 30 = F + HE 30; HE 100 = F + HE 100

All values are represented as mean ± SEM; n = 5; * = p < 0.05 control vs. treatment; & = p < 0.05 F vs. treatment

Conclusions

The ethanol extract of H. excelsa showed nephro-

protective and hepatoprotective effects by decreasing

of arterial hypertension, dyslipidaemia, proteinuria

and the expression of TGF-β1.

Acknowledgement

This work was supported by National Council for

Science and Technology (México) and Mexican

Council of Science and Technology (2016), fellowship

to Elizabeth Alejandrina Guzmán Hernández as part

of his post-doctoral research.


1118

Conflict of interest

The authors declare no conflict of interest.

References

1. McArdle MA, Finucane OM, Connaughton RM,

McMorrow AM, Roche HM, Mechanisms of obesity-

induced inflammation and insulin resistance: Insights

into the emerging role of nutritional strategies. Front

Endocrinol., 2013; 4: 52: 1-23.

2. Pommer W, Preventive Nephrology: The Role of

Obesity in Different Stages of Chronic Kidney Disease.

Kidney Dis (Basel), 2018; 4(4): 199-204.

3. Pashova-Stoyanova L, Tolekova A, Ganeva M,

Tsokeva Z, Hadzhibozheva P, Georgiev T, Nancheva

K, Vitamin D effects on lipid profile and uric acid

levels in the experimental model of metabolic

disorders in fructose fed wistar rats. Farmacia, 2019;

67(6): 1071-1076.

4. Navarrete A, Trejo-Miranda JL, Reyes-Trejo L, Principles

of root bark of Hippocratea excelsa (Hippocrataceae)

with gastroprotective activity. J Ethnopharmacol.,

2002; 79(3): 383-388.

5. Pérez RM, Pérez S, Zavala MA, Salazar M, Anti-

inflammatory activity of the bark of Hippocratea

excelsa. J Ethnopharmacol., 1995; 47(2): 85-90.

6. Kim DO, Jeong SW, Lee CY, Antioxidant capacity

of phenolic phytochemicals from various cultivars

of plums. Food Chem., 2003; 81: 321-326.

7. Mensor LL, Menezes FS, Leitao GG, Reis AS, Dos

Santos TC, Coube CS, Leitao SG, Screening of

Brazilian plant extracts for antioxidant activity by

the use of DPPH free radical method. Phytother Res.,

2001; 15: 127-130.

8. Miller NJ, Castelluccio C, Tijburg L, Rice-Evans C,

The antioxidant properties of theaflavins and their

gallate esters--radical scavengers or metal chelators?.

FEBS Lett., 1996; 392(1): 40-44.

9. Landry ML, Stanat S, Biron K, Brambilla D, Britt W,

Jokela J, Chou S, Drew WL, Erice A, Gilliam B,

Lurain N, Manischewitz J, Miner R, Nokta M,

Reichelderfer P, Spector S, Weinberg A, Yen-

Lieberman B, Crumpacker C, A standardized plaque

reduction assay for determination of drug susceptibilities

of cytomegalovirus clinical isolates. Antimicrob Agents

Chemother., 2000; 44(3): 688-692.

10. Merino-Aguilar H, Arrieta-Baez D, Jiménez-Estrada M,

Magos-Guerrero G, Hernández-Bautista RJ, Susunaga-

Notario Adel C, Almanza-Pérez, JC, Blancas-Flores

G, Román-Ramos R, Alarcón-Aguilar FJ, Effect of

fructooligosaccharides fraction from Psacalium

decompositum on inflammation and dyslipidemia in

rats with fructose-induced obesity. Nutrients, 2014;

6(2): 591-604.

11. Ku HK, Lim HM, Oh KH, Yang HJ, Jeong JS, Kim

SK, Interpretation of protein quantitation using the

Bradford assay: comparison with two calculation

models. Anal Biochem., 2013; 434(1): 178-180.

12. Guzmán-Hernández EA, Villalobos-Molina R, Sánchez-

Mendoza MA, Del Valle-Mondragón L, Pastelín-

Hernández G, Ibarra-Barajas M, Early co-expression

of cyclooxygenase-2 and renin in the rat kidney

cortex contributes to the development of N (G)-

nitro-L-arginine methyl ester induced hypertension.

Can J Physiol Pharmacol., 2015; 93(4): 299-308.

13. Ajiboye TO, Raji HO, Adeleye AO, Adigun NS,

Giwa OB, Ojewuyi OB, Oladiji AT, Hibiscus sabdariffa

calyx palliates insulin resistance, hyperglycemia,

dyslipidemia and oxidative rout in fructose-induced

metabolic syndrome rats. J Sci Food Agric., 2016;

96(5): 1522-1531.

14. Weiland F, Verspohl EJ, Local formation of angiotensin

peptides with paracrine activity by adipocytes. J Pept

Sci., 2009; 15(11): 767-776.

15. Tenorio-López FA, Zarco-Olvera G, Sánchez-Mendoza

A, Rosas-Peralta M, Pastelín-Hernández G, del Valle-

Mondragón L, Simultaneous determination of angiotensins

II and 1-7 by capillary zone electrophoresis in plasma

and urine from hypertensive rats. Talanta, 2010; 80(5):

1702-1712.

16. Nair AR, Elks CM, Vila J, Del Piero F, Paulsen DB,

Francis J, A blueberry-enriched diet improves renal

function and reduces oxidative stress in metabolic

syndrome animals: potential mechanism of TLR4-

MAPK signaling pathway. PLoS One, 2014; 9(11):

e111976: 1-12.

17. Aebi H, Catalase in vitro. Methods Enzymol., 1984;

105: 121-126.

18. Lawrence RA, Burk RF, Glutathione peroxidase

activity in selenium deficient rat liver. Biochem Biophys

Res Commun., 1976; 71(4): 952-958.

19. Sun Y, Oberley LW, Li Y, A simple method for

clinical assay of superoxide dismutase. Clin Chem.,

1988; 34(3): 497-500.

20. Yang M, Liu C, Jiang J, Zuo G, Lin X, Yamahara J,

Wang J, Li Y, Ginger extract diminishes chronic

fructose consumption-induced kidney injury through

suppression of renal overexpression of proinflammatory

cytokines in rats. BMC Complement Altern Med.,

2014; 14: 174: 1-12.

21. Novelli EL, Diniz YS, Galhardi CM, Ebaid GM,

Rodrigues HG, Mani F, Fernandes AA, Cicogna

AC, Novelli Filho JL, Anthropometrical parameters

and markers of obesity in rats. Lab Animals, 2007;

41(1): 111-119.

22. Mamikutty N, Thent ZC, Sapri SR, Sahruddin NN,

Mohd Yusof MR, Haji Suhaimi F, The establishment

of metabolic syndrome model by induction of fructose

drinking water in male Wistar rats. Biomed Res Int.,

2014; 2014: 263897: 1-8.

23. Uchida T, Shimizu M, Sakai Y, Nakano T, Hara K,

Takebayashi K, Inoue T, Node K, Inukai T, Takayanagi

K, Aso Y, Effects of losartan on serum total and

high-molecular weight adiponectin concentrations

in hypertensive patients with metabolic syndrome.

Metabolism, 2008; 57(9): 1278-1285.

24. Sharieh Hosseini SG, Khatamsaz S, Shariati M, The

effects of losartan on memory performance and leptin

resistance induced by obesity and high-fat diet in adult

male rats. Iran J Basic Med Sci., 2014; 17(1): 41-48.

25. Niranjana Murthy H, Dandin VS, Yoeup Paek K,

Hepatoprotective activity of ginsenosides from Panax

ginseng adventitious roots against carbon tetrachloride

treated hepatic injury in rats. J Ethnopharmacol., 2014;

158(Part A): 442-446.

26. Bulboacă A, Bolboacă SD, Suci S, Protective effect

of curcumin in fructose-induced metabolic syndrome


1119

and in streptozotocin-induced diabetes in rats. Iran

J Basic Med Sci., 2016; 19(6): 585-593.

27. Singh D, Arya PV, Sharma A, Dobhal MP, Gupta

RS, Modulatory potential of α-amyrin against hepatic

oxidative stress through antioxidant status in Wistar

albino rats. J Ethnopharmacol., 2015; 161: 186-193.

28. Prabhakar P, Reeta KH, Maulik SK, Dinda AK,

Gupta YK, α-Amyrin attenuates high fructose diet-

induced metabolic syndrome in rats. Appl Physiol

Nutr Metab., 2017; 42(1): 23-32.

29. Bezerra Carvalho KMM, de Melo TS, de Melo KM,

Gomes Quinderé AL, Bandeira de Oliveira FT,

Custódio Viana AFS, Gomes Nunes PI, da Silva

Quetz J, de Araújo Viana D, de Carvalho Almeida

da Silva AA, Havt A, da Cruz Fonseca SG, Chaves

MH, Amyrins from Protium heptaphyllum Reduce High-

Fat Diet-Induced Obesity in Mice via Modulation of

Enzymatic, Hormonal and Inflammatory Responses.

Planta Med., 2017; 83(03/04): 285-291.

30. Zhang DM, Jiao RQ, Kong LD, High Dietary Fructose:

Direct or Indirect Dangerous Factors Disturbing Tissue

and Organ Functions. Nutrients, 2017; 9(4): 335: 1-25.

31. Parhofer KG, Increasing HDL-cholesterol and prevention

of atherosclerosis: A critical perspective. Atheroscler

Suppl., 2015; 18: 109-111.

32. Rabie EM, Heeba GH, Abouzied MM, Khalifa MM,

Comparative effects of Aliskiren and Telmisartan

in high fructose diet-induced metabolic syndrome

in rats. Eur J Pharmacol., 2015; 760: 145-153.

33. Gersch MS, Mu W, Cirillo P, Reungjui S, Zhang L,

Roncal C, Sautin YY, Johnson RJ, Nakagawa T,

Fructose, but not dextrose, accelerates the progression

of chronic kidney disease. Am J Physiol Renal Physiol.,

2007; 293(4): F1256-1261.

34. Cho AS, Jeon SM, Kim MJ, Yeo J, Seo KI, Choi

MS, Lee MK, Chlorogenic acid exhibits anti-obesity

property and improves lipid metabolism in high-fat

diet-induced-obese mice. Food Chem Toxicol., 2010;

48(3): 937-943.

35. Mamikutty N, Thent ZC, Sapri SR, Sahruddin NN,

Mohd Yusof MR, Haji Suhaimi F, The establishment

of metabolic syndrome model by induction of fructose

drinking water in male Wistar rats. Biomed Res Int.,

2014; 2014: 263897: 1-8.

36. Kawarazaki W, Fujita T, The Role of Aldosterone

in Obesity-Related Hypertension. Am J Hypertens.,

2016; 29(4): 415-423.

37. Novelli EL, Diniz YS, Galhardi CM, Ebaid GM,

Rodrigues HG, Mani F, Fernandes AA, Cicogna AC,

Novelli Filho JL, Anthropometrical parameters and

markers of obesity in rats. Lab Animals, 2007;

41(1): 111-119.

38. El-Fawal R, El Fayoumi HM, Mahmoud MF, Diosmin

and crocin alleviate nephropathy in metabolic syndrome

rat model: Effect on oxidative stress and low grade

inflammation. Biomed Pharmacother., 2018; 102:

930-937.

39. Jefferson A, Shankland Sj, Pichler RH, Proteinuria

in diabetic kidney disease: a mechanistic viewpoint.

Kidney Int., 2008; 74(1): 22-36.

40. Ghayur A, Liu L, Kolb M, Chawla A, Lambe S,

Kapoor A, Margetts PJ, Adenovirus-mediated gene

transfer of TGF-beta1 to the renal glomeruli leads to

proteinuria. Am J Pathol., 2012; 180(3): 940-951.

41. Cabandugama PK, Gardner MJ, Sowers JR, The Renin

Angiotensin Aldosterone System in Obesity and

Hypertension: Roles in the Cardiorenal Metabolic

Syndrome. Med Clin North Am., 2017; 101(1): 129-137.

42. Chou CL, Lin H, Chen JS, Fang TC, Renin inhibition

improves metabolic syndrome, and reduces angiotensin

II levels and oxidative stress in visceral fat tissues

in fructose-fed rats. PLoS One, 2017; 10; 12(7):

e0180712: 1-17.

43. Rosenbloom J, Castro S, Jimenez SA, Narrative review:

fibrotic diseases: cellular and molecular mechanisms

and novel therapies. Ann Intern Med., 2010; 152:

159-166.

44. Watanabe D, Tanabe A, Naruse M, Morikawa S,

Ezaki T, Takano K, Renoprotective effects of an

angiotensin II receptor blocker in experimental model

rats with hypertension and metabolic disorders.

Hypertens Res., 2009; 32(9): 807-815.

45. Kong X, Zhang DY, Wu HB, Li FX, Losartan and

pioglitazone ameliorate nephropathy in experimental

metabolic syndrome rats. Biol Pharm Bull., 2011;

34(5): 693-699.

46. Yoshiji H, Fukui H, Renin-angiotensin system and

progression of chronic liver diseases. J Gastroenterol.,

2006; 41(10): 1020-1022.

47. Sies H, Oxidative stress: oxidants and antioxidants.

Exp Physiol., 1997; 82(2): 291-295.

48. Singal PK, Khaper N, Palace V, Kumar D, The role

of oxidative stress in the genesis of heart disease.

Cardiovasc Res., 1998; 40(3): 426-432.

49. Pérez-Barrón G, Avila-Acevedo JG, García-Bores

AM, Montes S, García-Jiménez S, León-Rivera I,

Rubio-Osornio M, Monroy-Noyola A, Neuroprotective

effect of Buddleja cordata methanolic extract in the

1-methyl-4-phenylpyridinium Parkinson's disease

rat model. J Nat Med., 2015; 69(1): 86-93.

50. Ávila Acevedo JG, Espinosa González AM, De Maria

y Campos DM, Benítez Flores J del C, Hernández

Delgado T, Flores Maya S, Campos Contreras J, Muñoz

López JL, García Bores AM, Photoprotection of

Buddleja cordata extract against UVB-induced skin

damage in SKH-1 hairless mice. BMC Complement

Altern Med., 2014; 14: 281: 1-9.

51. Biba Vikas, Akhil B S, Remani P, Sujathan K, Free

Radical Scavenging Properties of Annona squamosa.

Asian Pac J Cancer Prev., 2017; 18(10): 2725-2732.

52. Chung S, Yoon HE, Kim SJ, Kim SJ, Koh ES, Hong

YA, Park CW, Chang YS, Shin SJ, Oleanolic acid

attenuates renal fibrosis in mice with unilateral ureteral

obstruction via facilitating nuclear translocation of

Nrf2. Nutr Metab Lon., 2014; 11(1): 2: 1-11.

53. Son E, Yoon JM, An BJ, Lee YM, Cha J, Chi GY, Kim

DS, Comparison among Activities and Isoflavonoids

from Pueraria thunbergiana Aerial Parts and Root.

Molecules, 2019; 24(5): 912: 1-12.

54. Chung S, Yoon HE, Kim SJ, Kim SJ, Koh ES, Hong

YA, Park CW, Chang YS, Shin SJ, Oleanolic acid

attenuates renal fibrosis in mice with unilateral ureteral

obstruction via facilitating nuclear translocation of

Nrf2. Nutr Metab Lon., 2014; 11(1): 2: 1-11.

original article renoprotective and …€¦ · după tratarea timp de 6 săptămâni cu he (30 și...

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